Background

Alzheimer’s disease (AD) is characterized by amyloid deposition, tangle formation as well as synapse loss. Synaptic abnormalities occur early in the pathogenesis of AD. Identifying early synaptic abnormalities and their underlying mechanisms is likely important for the prevention and treatment of AD.

Methods

We performed in vivo two-photon calcium imaging to examine the activities of somas, dendrites and dendritic spines of layer 2/3 pyramidal neurons in the primary motor cortex in the APPswe/PS1dE9 mouse model of AD and age-matched wild type control mice. We also performed calcium imaging to determine the effect of Aβ oligomers on dendritic calcium activity. In addition, structural and functional two-photon imaging were used to examine the link between abnormal dendritic calcium activity and changes in dendritic spine size in the AD mouse model.

Results

We found that somatic calcium activities of layer 2/3 neurons were significantly lower in the primary motor cortex of 3-month-old APPswe/PS1dE9 mice than in wild type mice during quiet resting, but not during running on a treadmill. Notably, a significantly larger fraction of apical dendrites of layer 2/3 pyramidal neurons showed calcium transients with abnormally long duration and high peak amplitudes during treadmill running in AD mice. Administration of Aβ oligomers into the brain of wild type mice also induced abnormal dendritic calcium transients during running. Furthermore, we found that the activity and size of dendritic spines were significantly reduced on dendritic branches with abnormally prolonged dendritic calcium transients in AD mice.

Conclusion

Our findings show that abnormal dendritic calcium transients and synaptic depotentiation occur before amyloid plaque formation in the motor cortex of the APPswe/PS1dE9 mouse model of AD. Dendritic calcium transients with abnormally long durations and high amplitudes could be induced by soluble Aβ oligomers and contribute to synaptic deficits in the early pathogenesis of AD.

Amyloid plaques, neurofibrillary tangles and synapse loss are the pathological hallmarks of Alzheimer’s disease (AD) [1, 2]. AD patients exhibit progressive cognitive impairments, memory deficits and dementia. Previous studies have shown that synapse loss occurs many years before dementia and is the best correlate of cognitive impairment in AD patients [1, 3–6]. A significant reduction in postsynaptic spine density is detected before β-amyloid deposition in Tg2576 APP and PDAPP mouse models of AD [7–9]. Furthermore, down-regulation of various synaptic proteins has been observed in both AD patients and mouse models of AD [7, 10, 11]. These various synaptic deficits may underlie memory loss [8, 12, 13], as well as changes of sensory and motor systems observed at the early stage of AD [14–16].

The mechanisms underlying synaptic abnormalities and loss in AD remain unclear. Recent studies have shown that abnormal neuronal activities occur early in hippocampal and cortical regions in AD patients as well as in animal models of AD [8, 17–19]. For example, hyperactive neurons have been observed in the hippocampal CA1 region in 1.5 month-old APP23xPS45 Tg mice [19]. It has also been reported that the number of hypoactive and hyperactive cells in layer 2/3 neocortex increase in AD mice as compared to wild type mice [20]. Given the important role of neuronal activities in regulating synaptic plasticity [21, 22], alterations in neuronal activities likely cause abnormal synaptic structure and function in AD. However, the potential link between neuronal activity and synaptic abnormalities during the pathogenesis of AD remains elusive.

Many lines of evidence strongly suggest that soluble Aβ oligomers are the most neurotoxic Aβ peptides with detrimental effects on neurons and synapses in AD [17, 23–28]. The level of Aβ peptides, but not amyloid plaques, is the earliest known marker of AD and correlates with disease severity in patients [29–33]. Exogenous Aβ application into wild type mice can induce neuronal hyperactivity [34]. Furthermore, Aβ oligomers have been shown to disrupt calcium homeostasis in neurons by interacting with NMDA receptors, reducing glutamate uptake at synapses, and regulating calcium release from ER [35–41]. Because calcium elevation is an important trigger for synaptic plasticity [42, 43], these findings suggest that soluble Aβ oligomers may cause early synaptic deficits by affecting neuronal activity and calcium homeostasis in AD.

To better understand early synaptic deficits in AD, we performed transcranial two-photon calcium imaging of layer 2/3 neuronal somas, dendrites and dendritic spines in the motor cortex of 3-month-old APPswe/PS1dE9 transgenic mice before amyloid plaque formation [44–47]. We found that a fraction of dendritic branches of layer 2/3 pyramidal neurons exhibited a significant increase in the duration and amplitude of dendritic calcium transients during treadmill running in 3-month-old AD mice as compared with age-matched wild type control mice. Exogenous application of soluble Aβ oligomers into wild type mouse brain also increased the duration and peak amplitude of dendritic calcium transients during treadmill running. Notably, dendritic spines exhibited a decrease in activity and size after abnormal long-duration dendritic calcium transients. Together, these findings suggest that abnormal dendritic calcium elevation and synaptic depotentiation represent early dendritic and synaptic deficits in the pathogenesis of AD.

Experimental animals

The APPswe/PS1dE9 mice were purchased from Guangdong Medical Laboratory Animal Center, China. They overexpressed the Swedish mutation of APP, together with PS1 deleted in exon 9. Three-month-old mice were used in all the experiments. Mice expressing GCaMP6s in layer 2/3 and layer 5 pyramidal neurons were generated under the Thy-1 promoter in the Gan lab. Age-matched non-transgenic littermates served as controls. Mice of both sexes were used in the experiments. All experimental protocols were conducted in accordance with the institutional guidelines.

Administration of soluble Aβ42 oligomers or monomers

Aβ peptides were synthesized and provided by Dr. Zachary Wills from University of Pittsburgh. High performance liquid chromatography (HPLC), size exclusion chromatography (SEC) and electron microscopy (EM) were used to determine the concentration and oligomeric status of Aβ peptides as described in a recent publication [48]. Synthesized Aβ42 was dissolved in DMSO and aliquoted before freezing at −80 °C. 24 h prior to the experiment, Aβ42 was thawed and vortexed for 30 s to initiate the oligomerization and then incubated at 4 °C overnight. Before use, Aβ oligomers were diluted with artificial cerebrospinal fluid (ACSF) and used within 48 h of preparation at the concentration of 20 μM. 0.5 μl solution of Aβ42 oligomers (or Aβ42 monomers and vehicle) were slowly injected into the cortex of WT mice over 50 min. The vehicle or Aβ42 monomer without overnight incubation at 4 °C was used as control groups. To assess the global effect of Aβ42, we performed calcium imaging of neurons ~ 1 h after Aβ42 injection in the motor cortex contralateral to the injection site.

MK801 application

For local application of the NMDA receptor antagonist, MK801 (200 μM in artificial cerebrospinal fluid (ACSF), M107, Sigma-Aldrich), a glass microelectrode with a 20-μm outer diameter was inserted through a bone flap into the superficial layer of the cortex (20–30 μm below the pial surface) with an angle of 45° toward and ~100 μm away from the imaging area. The bone flap (~50 μm in diameter) for drug delivery was made adjacent to a thinned skull window for imaging.

Treadmill running

Mice were subjected to treadmill running with the same method as described in previous studies [43, 49]. Briefly, a custom built free-floating treadmill (101 cm × 58 cm × 44 cm) was used for motor training under a two-photon microscope. This free-floating treadmill allowed head-fixed mice to move their forelimbs freely to perform motor running tasks. To minimize motion artifact during imaging, the treadmill was constructed so that all the moving parts (motor, belt and drive shaft) were isolated from the microscope stage and the supporting air-table. Animals were positioned on a custom-made head-holder device that allowed micro-metal bars to be mounted. At the onset of a running trial, the treadmill motor driven by a DC power supply was turned on and the belt speed gradually increased from 0 cm/s to 8 cm/s within ~3 s. The speed of 8 cm/s was maintained for the rest of the trial. Each mouse was trained for 6–7 trials (5 min running and 1 min resting for each trial).

Surgery for two-photon imaging

Surgical procedures were performed similarly as described in previous studies [43, 50]. 24 h before imaging, surgery was performed to attach a head holder and to create a thinned-skull cranial window. First, mice were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (80 mg/kg). The mouse head was shaved and the skull surface was exposed with a midline scalp incision. The periosteum tissue over the skull surface was removed without damaging the temporal and occipital muscles. A head holder composed of two parallel micro-metal bars was attached to the animal’s skull to help restrain the animal’s head and reduce motion-induced artifact during imaging. A small skull region (~0.2 mm in diameter) was located over the primary motor cortex based on stereotaxic coordinates [51] (1.0 mm posterior from bregma and 1.5 mm lateral from the midline) and marked with a pencil. A thin layer of cyanoacrylate-based glue was first applied to the top of entire skull surface, and the head holder was then mounted on top of the skull with dental acrylic cement such that the marked skull region was exposed between the two bars. Precaution was taken not to cover the marked region with dental acrylic cement.

Before imaging of dendrites and dendritic spines, a high-speed micro-drill and microsurgical blade were used to thin a circular area over the marked region to a thickness of approximately 20 μm. The thinning procedure generally took less than 5 min.

To image somas at the depth of ~250 μm below the pial surface, the skull overlying the motor cortex of interest (1 mm × 1 mm) was removed and replaced with a glass window right before two-photon imaging as described previously [43].

Two-photon imaging of somas, dendrites and dendritic spines in the motor cortex

Genetically encoded calcium indicator GCaMP6s was used for calcium imaging in the primary motor cortex of awake, head-restrained mice. For calcium imaging of APPswe/PS1dE9 mice and wild type controls, a total amount of 0.1–0.2 μl of AAV-synapsin-GCaMP6s (AAV serotype 2/1; >2 × 1013 (GC/ml) titer; from the University of Pennsylvania Gene Therapy Program Vector Core) was diluted two times in ACSF and slowly injected (Picospritzer III; 20 p.s.i., 20 ms, 0.3 Hz) over 10–15 min into layer 2/3 of the motor cortex (0–1.5 mm posterior from bregma and 0–1.5 mm lateral from midline) using a glass microelectrode. Mice were prepared for calcium imaging ~18 days after virus injection.

For calcium imaging of mice injected with Aβ42 oligomers and monomers, we used wild type mice infected with AAV-synapsin-GCaMP6s, as well as transgenic mice expressing GCaMP6s in layer 2/3 and layer 5 pyramidal neurons under the Thy-1 promoter. This transgenic mouse line was generated in the Gan lab. Calcium imaging of dendrites and somas was performed in mice immediately after Aβ42 injection.

For simultaneous calcium and structural imaging, three viruses (AAV1.hSyn.Cre.WPRE.hGH, AAV1.CAG.Flex.tdTomato.WPRE.bGH and AAV1.CAG.Flex.GCaMP6s.WPRE.SV40 from the University of Pennsylvania Gene Therapy Program Vector Core) were mixed at 1:15:80 volume ratios, and ~0.1 μl of the three mixed viruses were injected into layer 2/3 of the primary motor cortex. Viruses were injected into the primary motor cortex ~18 days before calcium and dendritic spine imaging.

To image somas at the depth of ~250 μm below the pial surface, the skull overlying the motor cortex of interest (1 mm × 1 mm) was removed and replaced with a glass window right before two-photon imaging. Dendritic calcium transients and spine calcium transients were imaged at the depth of ~50 μm below the pial surface through a thinned skull window. Neuronal somas were imaged at the depth of ~250 μm below the pial surface via a glass window. The imaging window was immersed in ACSF and the head-restrained animal was placed on the stage of a two-photon laser scanning microscopy (Olympus Fluoview 1000 two-photon system equipped with MaiTai DeepSee Ti:Sapphire laser from Spectra Physics). The laser was tuned to the wavelength of 920 nm with laser power of ~ 20 mW on the tissue sample. Calcium signals were recorded at 2 Hz using a 25X objective (N.A. 1.05, 3X digital zoom). Dendritic spine structures were imaged at 0 h and 1.5 h using a 25X objective (N.A. 1.05, 3X digital zoom).

During quiet resting state, we took images for 60 s. During running, we acquired images for 5 running trials. Each running trial lasted 60 s. After the completion of each trial, the treadmill was turned off and the next trial started after a 60-s rest period.

Image analysis

Neuronal, dendritic and spine calcium activity, indicated by GCaMP6 fluorescence changes, were analyzed post hoc using Image J software (NIH). The GCaMP6 fluorescence (F) during quiet resting and running was measured by averaging pixels within each identifiable area (Regions of interests, ROIs). Then ∆F/F0 was calculated as ∆F/F0 = (F-F0)/ F0, in which ∆F was F-F0 (all F values were subtracted a background fluorescence value of vessels), and F0 was the average of 10% minimum F values over 1 min period, representing baseline fluorescence. The threshold for determining calcium transients was calculated as three times the standard deviation (SD) of baseline fluorescence. The frequency of calcium transients was calculated as the number of calcium transients per minute on each dendrite. The duration was calculated as the total time of calcium transients with ∆F/F0 above the threshold. The peak amplitude was the highest value of the calcium transient. The total calcium activity was the accumulated calcium activities above the threshold per minute.

Similar to previous studies [42], back-propagating action potential-related calcium component was removed from spine calcium signals by subtracting a scaled version of the dendritic shaft signal, ΔF/F0spine_specific = ΔF/F0spine – 0.7 x ΔF/F0dendrite. We defined active spines as those with spine-specific calcium transients that crossed 3 SD of baseline fluorescence and inactive spines as those less than 3 SD.

Spine head size was measured according to previous studies [43, 49]. After background subtraction, the fluorescence intensity of tdTomato in the spine (the intensity of all pixels covering the spine in the best focal plane) was divided by the fluorescence intensity of the adjacent dendritic shaft. tdTomato fluorescence intensity of a spine was measured as follows, where Area was the number of pixels in an oval surrounding the head of the spine and mean optical density (Mean OD) was the mean brightness of pixels in that area:

After spine head sizes were measured on the same dendritic segment, spine size changes were calculated by comparing spine size measurement between imaging sessions.

Statistics

All data were presented as mean ± s.e.m. The Kolmogorov-Smirnov test was used to test whether datasets were normally distributed. If so, we used a two-tailed Student’s t test to compare two groups. If not, tests for differences between groups were performed using non-parametric tests. Two Independent-samples Mann-Whitney Test and Related-samples Wilcoxon Signed Rank Test ware used to compare differences between groups whose distributions did not pass Kolmogorov-Smirnov test. Significant levels were set at p ≤ 0.05. All statistical analyses were performed using the IBM SPSS Statistics 23.

When mice were subjected to treadmill running, the frequency of spine calcium transients was significantly higher in AD mice as compared to that in WT mice (Fig. 2g; WT: n = 33 spines from 4 mice; AD: n = 34 spines from 5 mice. P = 0.002). The duration, peak amplitude and total integrated activity of spine calcium transients were comparable between AD and WT mice (Fig. 2h - j; P > 0.3). Together, these results indicate that the overall level of synaptic activity is comparable between WT and AD mice under both quiet resting and running conditions.

The duration and peak ΔF/F0 of dendritic calcium transients are significantly higher during running in AD mice than in WT mice

Many lines of evidence indicate that dendritic calcium spikes are critical for synaptic plasticity of excitatory neurons [52–57]. Recent studies have shown that dendritic calcium spikes in apical dendrites of layer 5 pyramidal neurons are important for dendritic spine plasticity in the motor cortex [43]. To investigate potential dendritic deficits in AD mice, we examined dendritic calcium activity of L2/3 pyramidal neurons in WT and AD mice under both quiet resting and treadmill running conditions.

In the superficial layer of the motor cortex, we observed large dendritic calcium transients that occurred across long segments (> 30 μm) of apical tuft dendrites of layer 2/3 pyramidal neurons (Fig. 3a-c). The vast majority of these dendritic calcium transients lasted more than hundreds of milliseconds and had comparable ΔF/F0 across long stretches of dendrites, resembling NMDAR-activation-dependent dendritic calcium spikes described previously [43, 58–60] (Fig. 3c). Under the quiet resting condition, we found that the frequency and duration of such dendritic calcium transients were not significantly different between WT and AD mice (Fig. 3d, e; WT: n = 90 calcium transients from 4 mice; AD: n = 121 calcium transients from 5 mice; P > 0.05). However, the peak ΔF/F0 of dendritic calcium transients was significantly higher in AD mice than in WT mice (Fig. 3f; P = 0.005).

Many lines of evidence indicate that soluble Aβ42 oligomers can alter neuronal activity and lead to synaptic loss [61–65]. To investigate the effect of Aβ oligomers on neuronal activities in vivo, we injected Aβ oligomers into the motor cortex of wild type mice infected with AAV-hSyn-GCaMP6s virus. One hour after the injection of Aβ oligomers, we performed calcium imaging of somas, spines and apical dendrites of pyramidal neurons in the M1 contralateral to the site of Aβ injection (Fig. 4a-c). During treadmill running, we found that Aβ oligomers induced significantly higher somatic calcium activities (Additional file 1: Figure S1) but had no significant effect on the overall level of spine calcium activities of layer 2/3 pyramidal neurons (Additional file 2: Figure S2). Notably, while the frequency of dendritic calcium transients was not significantly different between vehicle and Aβ oligomer-injected mice (Fig. 4d; Vehicle: n = 136 calcium transients from 4 mice; Aβ42 oligomer: n = 99 calcium transients from 4 mice; P = 0.034), the injection of Aβ oligomers caused a significantly longer duration and higher peak amplitude of dendritic calcium transients as compared to vehicle injection (Fig. 4e-f; P < 0.05). These findings indicate that during treadmill running, Aβ oligomers cause a rapid increase in neuronal somatic calcium activities, as well as abnormally long duration of dendritic calcium transients in layer 2/3 neurons of the motor cortex.

The size of dendritic spines associated with long-duration dendritic calcium transients is reduced in AD mice

To further investigate the impact of long-duration dendritic calcium transients in AD mice, we examined changes in dendritic spine number and size on apical tuft dendrites of L2/3 pyramidal neurons expressing both GCaMP6s and a structural maker, tdTomato, in the M1 region (Fig. 6a). In this experiment, we first imaged dendritic spines at 0 h, trained mice on the treadmill for ~1.5 h, and performed imaging again at 1.5 h. Over the period of 1.5 h running, we found no formation or elimination of dendritic spines in both WT and AD mice (Additional file 3: Figure S3: 72 and 89 spines examined in WT and AD mice, respectively).

In this study, we report that before amyloid plaque deposition, dendritic calcium transients with abnormally long durations and high peak amplitudes occur on apical dendrites of layer 2/3 pyramidal neurons in the motor cortex of 3 month-old APPswe/PS1dE9 transgenic mice. These abnormally long dendritic calcium transients could be induced after administration of soluble Aβ oligomers. Furthermore, long-duration dendritic calcium transients are associated with the reduction of dendritic spine calcium peak amplitude and spine size. Together, these results show that prolonged dendritic calcium transients and increased synaptic depotentiation are early neuronal deficits that occur before amyloid plaque formation in AD mice.

Previous studies have shown that L2/3 cortical neurons exhibit both hyperactivity and hypoactivity in aged APP23 × PS45 mice [20]. These hyperactive neurons are mostly clustered around amyloid plaques, suggesting that the microenvironment near amyloid plaques play an important role in triggering hyperactivity of nearby neurons [20]. Our findings indicate that prior to amyloid plaque deposition, calcium activities of L2/3 neuronal somas during the quiet resting state are lower in 3-month-old APPswe/PS1dE9 AD mice than in WT mice. Furthermore, somatic calcium activities of these cells during treadmill running have no significant difference between AD and age-matched WT mice. Thus, unlike the hyperactivity of cortical neurons near amyloid plaques in aged APP23 × PS45 mice, there is no overall increase in the activities of cortical neurons in the plaque-free motor cortex of 3-month-old APPswe/PS1dE9 mice. It is worth to note that neuronal hyperactivity has been observed in the hippocampus of 1.5 month APP23 × PS45 mice before the formation of amyloid plaques [19]. Further studies are needed to investigate whether neuronal hyperactivity may occur in the hippocampus, but not in the motor cortex, of 3-month-old APPswe/PS1dE9 mice. If so, it would suggest that alterations of neuronal activities in AD mice occur at different stages in different brain regions.

One key finding of our study is that during the active running state, a fraction of apical dendrites of L2/3 pyramidal neurons exhibit dendritic calcium transients with abnormal long-duration and high-amplitude in the motor cortex of APPswe/PS1dE9 mice, prior to amyloid plaque formation. Recent studies have shown that dendritic calcium spikes are critical for changes of spine calcium activity and spine size in the motor cortex [43]. Consistent with the fundamental role of dendritic calcium elevation in synaptic plasticity, we found that peak amplitudes of dendritic spines decrease after prolonged dendritic calcium transients (> 8 s). Furthermore, long-duration dendritic calcium transients are associated with a reduction in the size of active spines, but not inactive spines, at the generation of dendritic calcium transients. Because calcium activities of neuronal somas were comparable between APPswe/PS1dE9 and WT mice during running, these results suggest the dysregulation of dendritic calcium and spine plasticity are among the earliest-occurring neuronal deficits (prior to the neuronal somatic hyperactivity and plaque formation) in the pathogenesis of AD.

We found that administration of Aβ oligomers in WT mice causes abnormal long-duration dendritic calcium transients. This finding is consistent with the detrimental roles of soluble Aβ oligomers in disrupting calcium homeostasis and synaptic plasticity in AD. Aβ oligomers and PS1 mutations can disrupt calcium release from ER [71–77]. Soluble Aβ oligomers can also disrupt dendritic calcium by interacting with NMDA receptors, modulating glutamate uptake and regulating calcium release from ER [35–41]. Such alterations of dendritic and spine calcium activity would likely have detrimental impacts on synaptic function and may lead to a progressive loss of synapses in AD. It is important to note that although we found synaptic depotentiation associated with prolonged dendritic calcium activity, we did not observe an increase of spine loss over 1.5 h of treadmill running. Future studies are therefore needed to investigate the relationship among long-duration dendritic calcium activity, spine depotentiation and potential spine elimination over extended periods of time to better understand synapse loss in AD pathogenesis.

Many lines of evidence indicate that NMDAR activation plays important roles in the generation of dendritic calcium spikes and synaptic plasticity [43, 58–60]. Consistent with these previous studies [43, 58–60], we observed that NMDAR antagonist MK801 application significantly reduced the frequency, duration and peak amplitude of dendritic and spine calcium transients (Additional file 4: Figure S4 a-f). Furthermore, after MK801 application, there were no significant changes in the peak amplitude of spine calcium transients before and after dendritic calcium transients (Additional file 4: Figure S4 g). These findings raise the possibility that the use of NMDAR antagonists such as memantine may slow down AD progression in part by reducing prolonged dendritic calcium activity and synaptic abnormalities. Future studies are needed to investigate whether memantine and other pharmacological agents targeting abnormal dendritic calcium activities and their signaling may help the treatment of AD by alleviating synaptic depotentiation and loss.

Our findings show that abnormal long-duration dendritic calcium transients and decreases in spine size occur prior to amyloid plaque formation and overall changes of neuronal somatic activity in the APPswe/PS1dE9 mouse model of AD. The abnormal long-duration and high-amplitude dendritic calcium transients can be induced by soluble Aβ oligomers and may contribute to synaptic deficits during the early pathogenesis of AD.

Acknowledgements

We thank all the members of Gan lab for critical comments and helpful discussions on this manuscript.

Funding

This work was supported by NIH 5R01 NS087198 and 5R01NS047325 to W.-B.G., Shenzhen Science and Technology Innovation Funds KQTD2015032709315529 and JSGG20140703163838793 to W. L., Shenzhen Science and Technology Innovation Funds JCYJ20160428154351820 to L.M., NIMH R21-MH107966 to Z.P.W.

Availability of data and materials

All data analyzed in this study are included within the article.

Authors’ contributions

YB and WBG designed the experiments and wrote the manuscript; YB, ML, YMZ and LM performed in vivo imaging experiments with the help of WL; WLH bred transgenic mice and performed genotyping; YB analyzed the data with the help of LM. ML, QQ and WL. All authors read and approved the final manuscript.

Authors’ information

Information for all the co-authors is listed in the title page.

Ethics approval and consent to participate

Animal studies with APP/PS1 mice were performed in compliance with the institutional guidelines at Peking University Shenzhen Graduate School. Animal studies with Aβ oligomers were performed in compliance with IACUC (Institutional Animal Care and Use Committee) at New York University School of Medicine.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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